This invention concerns cell and tissue growth substrates, growth stimulation compositions, and methods for delivering growth factors to cells and tissues.
Long-term mammalian cell culture has been difficult to achieve. Many types of specialized cells plated on standard tissue culture plastic dishes dedifferentiate, lose function, and fail to proliferate. There are many applications of mammalian cell culture that could benefit from methods or materials which enhance the long term stability of differentiated mammalian cells in culture. These cells are currently used as sources of natural and engineered proteins and glycoproteins, in screens for the effects of compounds on cell proliferation and function, and for implantation to supplement or replace cell function. Certain cells are particularly difficult to maintain in long term culture, such as hepatocytes.
It would be especially useful if hepatocytes could be maintained in long term culture. For example, in vitro toxicity testing of orally administered compounds has been hampered by the fact that the liver converts many compounds into other chemical forms. These other forms may be toxic or have other effects. Thus, complete testing of materials in cell culture must include the effects of biotransformations carried out by the liver. Using current methodology, it is difficult to grow normal liver cells in vitro beyond two to three cell divisions. The result is that in vitro testing does not reduce the number of animals needed because essentially all of the cells to be used in vitro must come from direct isolation. A method of expanding liver cells in vitro would make it feasible to use in vitro liver cell cultures to carry out biotransformations by applying the compound of interest directly to liver cells in culture. The supernate from the liver cell cultures could then be applied to other types of cells, such as skin, lung, nerve, and bladder, to assess the effect of the metabolized compound of interest.
Studies have been conducted for a number of years to improve the viability, proliferation and differentiated function of eukaryotic cells cultured in vitro. One discovery has been the importance of the extracellular matrix and extracellular matrix molecules in maintaining cell function and allowing cell growth. These effects, and methods of using matrix components for cell growth, have been described by, for example, Jauregui et al., In Vitro Cellular & Developmental Biology 22: 13-22 (1986), Kleinman et al., Analytical Biochemistry 166: 1-13 (1987), and Mooney et al., Journal of Cellular Physiology 151: 497-505 (1992).
Growth factors, such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and transforming growth factors (TGF.alpha., TGF.beta.), exert a broad mitogenic response. Growth factors and their effects have been described in "Peptide Growth Factors and Their Receptors I" M. B. Sporn and A. B. Roberts, eds. (Springer-Verlag, New York, 1990). In recognition of their importance, most cell and tissue growth compositions include growth factors, either as an additive or as a component of complex growth media. The use of growth factors in this manner has certain drawbacks. For example, cells have a complex, nonlinear response to the concentration of growth factor in their environment. Extended exposure to high growth factor concentrations may cause cells to lose responsiveness to the factor. For example, EGF, a potent mitogen for a wide variety of cell types and arguably the best-characterized of the growth factors, when delivered in soluble form, is typically internalized by the cell, and the cell often responds by down-regulating the number of EGF receptors. This down-regulation causes cells to lose responsiveness to EGF.
Growth factors have also been used in disappointingly few clinical products, considering the range of effects they produce in vitro. Translation of the mitogenic effects observed for the target cell in vitro to tissue growth in vivo is hampered by several issues. For example, the growth factors, when placed in a complex cellular environment, often end up stimulating the growth of competing cells which then overgrow the target cells. Researchers have attempted to solve this problem by targeting delivery of factors at a specific site, but this approach is not always successful because soluble growth factors can readily diffuse into the blood stream and away from the target site, exerting their effects elsewhere. This diffusion of growth factors is also a problem because it increases the amount of growth factor that must be used in order to have the desired local effect. Internalization of growth factors and loss of responsiveness to growth factors is a particular problem for in vivo applications considering the amount of time cell growth must be stimulated to allow wound healing.
Another attempt to improve the longevity of growth factor effects in vivo has been to incorporate growth factors in a slow release material. Such a scheme still requires large amounts of growth factor and does not address the problem of competing cell growth due to diffusion of the growth factors. The large amount of growth factors needed for these cell and tissue growth methods is a particular problem because growth factors are difficult and expensive to prepare.
It is therefore an object of the invention to provide a cell and tissue growth substrate that stimulates long-term target cell growth.
It is another object of the invention to provide a tissue growth scaffold for growth of a target tissue in vivo.
It is a further object of the invention to provide a method of long-term cell and tissue growth in vitro, and to provide a method of growing target tissue in vivo.
It is another object of the invention to provide an in vitro tissue analog for drug and toxicity testing, and a method of drug and toxicity testing using the tissue analog.